Signaling via heterotrimeric (henceforth trimeric) G proteins regulates most, if not all, physiological functions and its dysregulation is the cause of vast array of diseases. More than 30% of marketed drugs target G protein-coupled receptors (GPCRs), which are the classical activators of trimeric G proteins. We have recently identified a novel mechanism of signal transduction in which trimeric G proteins are activated by a cytoplasmic, non-receptor protein called GIV (aka Girdin) instead of by GPCRs. This mechanism has important biomedical implications because its upregulation is tightly associated with the development of metastasis. Despite that metastasis causes ~90% of cancer-related deaths, this process is still poorly understood and remains incurable. Thus, the existence of a novel, receptor-independent mechanism of G protein activation by GIV not only provides a new perspective on G protein signaling regulation but also opens new opportunities for pharmacological intervention. However, no existing chemical probe targets the GIV-G protein coupling. Our goal is to identify chemical probes that specifically inhibit the GIV-G protein interaction as research tools to further understand a novel mechanism of G protein signaling and its impact in disease. BACKGROUND: Previous work has established that a short linear motif of ~25 aa in GIV is responsible for its biological activity as a G protein activator (Guanine nucleotide Exchange Factor, GEF) and as an enhancer of prometastatic behavior in tumor cells. Our published and unpublished data provide a wealth of structural information indicating that the GIV-G protein interaction is a tractable molecular interface that can be specifically targeted. Taken together, these results provide the proof of principle to consider the GIV-G?i interface a bona fide molecular target in signaling and cancer metastasis. Our hypothesis is that chemical probes that specifically inhibit the GIV-G?i interface will be valuable research tools to investigate a novel mechanism of G protein activation during metastasis and will lay the foundation for future targeted therapies. RESEARCH PLAN: All the assays required for this campaign have been established in our hands, including validation of the screen/ counter-screen assays in a pilot screen (Z'e0.7). In SA#1 we will carry out a high throughput screen (HTS) of >150,000 compounds using a fluorescence polarization (FP). Hits will be tested in a counter-screen assay based on AlphaScreen(R) to remove false positives and will be triaged by quality control of compounds and medicinal chemistry assessments. In SA#2 we will perform follow-up assays in yeast and mammalian cells to assess efficacy, specificity and toxicity. Filtered compounds and analogs optimized for potency will be eventually tested for their ability to block tumor cell migration as a surrogate measure of the bioactivity of the target (GIV-G?i) in metastasis. The HTS and assay automations will be performed at the ICCB-Longwood screening facility (Harvard Medical School) and the medicinal chemistry efforts required to triage hits and synthesize analogs will be carried by the co-investigator Aaron Beeler (Boston University).